GENERAL OVERVIEW: The Flow Cytometry Core at NEI provides flow cytometry analytical and sorting equipment and services to the NEI Intramural community. It utilizes and develops state-of-the-art sample preparation, data acquisition and analysis, and sorting procedures in collaborative research projects. Provides training to students, fellows, and principal investigators on sample preparation, staining, and post-sort handling. Assesses technical research needs and recommends recruitment of the appropriate staff and acquisition of the equipment needed to meet those needs. The 2 sorters, FACS Aria II and FACS Aria Fusion, are largely operated by trained Core personnel. The analytical instruments, 2 MACS Quant instruments and the BD FACS Calibur, are largely operated by users who receive training from Core personnel. SERVICES PROVIDED THE CORE: This year, eighty individuals from thirteen different laboratories used the facility. These services and collaborative services were performed for 8 Principal Investigators (PIs) from 8 NEI labs (ERPD, LI, LRCMB, MSF, N-NRL, RN, OGVFB and UNGIRD), plus 3 PIs from 3 other institutes at NIH (NICHD, NIDCD and NIDDK). Over 10,000 samples were analyzed. This year the core performed about 1,000 hours of sorting. Among the techniques now in use within the core are methods for phenotyping live cells, detecting gene expression, monitoring membrane and DNA content changes due to apoptosis or proliferation, measurement of intracellular proteins and quantification of soluble proteins. The work involving human tissues includes the sorting of peripheral blood mononuclear cells to study their cytokine production, genotype and DNA or RNA expression. The sources are blood, buffy coat, and white cells. Some analytical work had been done with eye fluids, eye tissue specimen, protein, and tears. The National Eye Institute made a great investment in biosafety with the addition BD FACSAria Fusion flow cytometer equipped with a fully integrated biosafety cabinet. This sorter meets the recent NIH's operator and sample protection requirements as well as global standards for bioprotection for processing human samples. No human tissues were stored by the core. The Core encourages, but does not require, users to acknowledge the Core contribution in their publications. The following are examples of the publications that used NEI Flow Cytometry Core resources: Kim JW, Yang HJ, Brooks MJ, Zelinger L, Karaklah G, Gotoh N, Boleda A, Gieser L, Giuste F, Whitaker DT, Walton A, Villasmil R, Barb JJ, Munson PJ, Kaya KD, Chaitankar V, Cogliati T, Swaroop A. NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors. Cell Rep. 2016 Nov 22;17(9):2460-2473. doi: 10.1016/j.celrep.2016.10.074. Zaitseva E, Zaitsev E, Melikov K, Arakelyan A, Marin M, Villasmil R, Margolis LB, Melikyan GB, Chernomordik LV. Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine. Cell Host Microbe. 2017 Jul 12;22(1):99-110.e7. doi: 10.1016/j.chom.2017.06.012. St Leger AJ, Desai JV, Drummond RA, Kugadas A, Almaghrabi F, Silver P, Raychaudhuri K, Gadjeva M, Iwakura Y, Lionakis MS, Caspi RR. An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal T Cells. Immunity. 2017 Jul 18;47(1):148-158.e5. doi: 10.1016/j.immuni.2017.06.014. Epub 2017 Jul 11. Dambuza IM, He C, Choi, JK, Yu CR, Wang R, Mattapallil MJ, Wingfield PT, Caspi RR, and Egwuagu CE. (In Revision) IL-12p35 induces expansion of IL-10- and IL-35-expressing regulatory B cells (Bregs) and ameliorates autoimmune disease. Nature Communications (In Press). Choi JK, Dambuza IM, He C, Yu CR, Uche AN, Mattapallil MJ, Caspi RR, and Egwuagu CE. IL-12p35 Inhibits Neuroinflammation and Ameliorates Autoimmune Encephalomyelitis. Frontiers In Immunology (In Press) He C, Yu CR, Mattapallil MJ, Sun L, Larkin J 3rd, Egwuagu CE. (2016). SOCS1-mimetic Peptide Suppresses Chronic Intraocular Inflammatory Disease (Uveitis). Mediators Inflamm. 2016:2939370. Sun L, St Leger AJ, Yu CR, He C, Mahdi RM, Chan CC, Wang H, Morse HC 3rd, Egwuagu CE. (2016) Interferon Regulator Factor 8 (IRF8) Limits Ocular Pathology during HSV-1 Infection by Restraining the Activation and Expansion of CD8+ T Cells.PLoS One. 11(5):e0155420. doi: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice. Wang X, Wei Y, Xiao H, Liu X, Zhang Y, Han G, Chen G, Hou C, Ma N, Shen B, Li Y, *Egwuagu CE, *Wang R. (2016) Eur J Immunol. 71:54-63. Wang X, Wei Y, Xiao H, Liu X, Zhang Y, Han G, Chen G, Hou C, Zhang L, Ma N, Shen B, Li Y, *Egwuagu CE, *Wang R. (2016). Pre-existing CD19-independent GL7- Breg cells are expanded during inflammation and in mice with lupus-like disease. Mol Immunol. 71: 54-63 Tan C1, Wandu WS1, Lee RS2, Hinshaw SH1, Klinman DM3, Wawrousek E2, Gery I4. Shedding New Light on the Process of Licensing for Pathogenicity by Th Lymphocytes. J Immunol. 2017 Jan 15;198(2):681-690. doi: 10.4049/jimmunol.1502108. Epub 2016 Dec 16. Mead B1, Tomarev S1. Bone Marrow-Derived Mesenchymal Stem Cells-Derived Exosomes Promote Survival of Retinal Ganglion Cells Through miRNA-Dependent Mechanisms. Stem Cells Transl Med. 2017 Apr;6(4):1273-1285. doi: 10.1002/sctm.16-0428. Epub 2017 Jan 26. The availability of the Technical IRTA position continues to have a very positive effect on the Core operation. There is a great need for well-trained flow cytometry operators. Through the TechIRTA, the hours of operation of the facility have been expanded from 40 hr/week to 60 hr/week. The expanded hours of operation helped to reduce the backlog on the sorter schedule. The TechIRTA position has greatly enhanced the ability of the Core to use the cell sorter for high throughput multicolor analysis. Several formal training sessions were offered by the core to the NEI community. These courses included: Introduction to Flow Cytometry, Advanced Techniques (Ex. Apoptosis applications), Analytical Instrument Operation and Data Analysis with FloJo Software. The Core Manager completed 40 hours of continuing education in management of core laboratories. The technical IRTA completed 20 hours of formal training outside the Institute.